calcimycin and sphingosine-1-phosphate

Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.

Topics: Biological Assay; Bradykinin; Calcimycin; Calcium; Calcium Ionophores; Calcium Signaling; Drug Discovery; Fluorescent Dyes; High-Throughput Screening Assays; Histamine; Humans; Kinetics; Lysophospholipids; Myocytes, Smooth Muscle; Primary Cell Culture; Reagent Kits, Diagnostic; Receptors, G-Protein-Coupled; Respiratory System; Sphingosine

Receptor-regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca(2+) response evoked by sphingosine 1-phosphate (S1P) also participates to the enzyme regulation. Ca(2+) was found critical for PKC(alpha)-mediated PLD activation. Moreover, S1P-induced PLD activity resulted diminished by calmodulin inhibitors such as W-7 and CGS9343B implicating its involvement in the process. A plausible candidate for Ca(2+)-dependent PLD regulation by S1P was represented by calcineurin, in view of the observed reduction of the stimulatory effect by cyclosporin A. In contrast, monomeric GTP-binding protein Ral was translocated to membranes by S1P in a Ca(2+)-independent manner, ruling out its possible role in agonist-mediated regulation of PLD.

Topics: Animals; Calcimycin; Calcium; Cell Line; Chelating Agents; Egtazic Acid; Ionophores; Isoenzymes; Lysophospholipids; Mice; Phospholipase D; Protein Kinase C; Protein Kinase C-alpha; Sphingosine

In this study, the ability of sphingosine-1-phosphate (S1P) to modulate platelet aggregation induced by other agents in platelet-rich plasma (PRP) was investigated. S1P alone did not stimulate platelet aggregation in PRP. S1P inhibited the platelet aggregation induced by the TRAP peptide (6.75 microM), noradrenaline (NA; 12.5 microM) and the Ca2+ ionophore (5.0-9.5 microM). S1P also increased the response time of platelets to arachidonic acid (AA), but decreased the response time to PMA. S1P displayed a dual effect on sodium fluoride (NaF)-induced platelet aggregation and had no effect on the aggregation induced by ADP or lysophosphatidic acid (LPA). Furthermore, S1P blocked the synergistic interaction of oleyol-lysophosphatidic acid (O-LPA) with the TRAP peptide or noradrenaline, while the synergistic interaction of O-LPA with ADP remained largely unaffected.

Topics: Adenosine Diphosphate; Calcimycin; Humans; Kinetics; Lysophospholipids; Male; Norepinephrine; Phosphatidic Acids; Platelet Aggregation; Platelet Aggregation Inhibitors; Proteins; Receptors, Thrombin; Sodium Fluoride; Sphingosine

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.

Topics: Animals; Apoptosis; Calcimycin; Calcium; Cell Line; Ceramides; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Activation; Fibroblasts; Genistein; Insulin; Insulin-Like Growth Factor I; Lysophospholipids; Mice; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Receptor, IGF Type 1; Signal Transduction; Sphingosine; Thapsigargin; Vanadates

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we showed in in vitro experiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP(2) concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca(2+) ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-alpha and PKC-beta1 but not PKC-epsilon was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from G(i) for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.

Topics: 3T3 Cells; Animals; Bradykinin; Calcimycin; Cell Membrane; DNA, Complementary; Egtazic Acid; Enzyme Activation; Gelsolin; Humans; Isoenzymes; Kinetics; Lysophospholipids; Mice; Mitogen-Activated Protein Kinases; Pertussis Toxin; Phosphatidylinositol 4,5-Diphosphate; Phospholipase D; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Kinase C-epsilon; Recombinant Proteins; Sphingosine; Tetradecanoylphorbol Acetate; Transfection; Type C Phospholipases; Virulence Factors, Bordetella

Sphingosine and sphingosine derivatives induce Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653-1656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of sphingosine and sphingosine derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT1MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Addition of 15 microM sphingosine to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80% of pumped Ca2+), but in contrast to sphingosine, there was no lag and the effect was independent of ATP or ADP. The EC50 for sphingosine and SPC in activating Ca2+ release was 1 and 3 microM, respectively. Such observations are consistent with the view that sphingosine, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. Sphingosine 1-phosphate (sph-1-P), the likely active Ca(2+)-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca(2+)-releasing activity. sph-1-P at 10 microM induced Ca2+ release from both B3 vesicles and permeabilized DDT1MF-2 cells to exactly the same extent as sphingosine. Unlike sphingosine, the effect of sph-1-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [gamma-32P]ATP and sphingosine under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-1-P. Formation of this labeled product was prevented by removal of exogenous sphingosine and blocked by ADP. Sphingosine- but not SPC-mediated Ca2+ release was blocked by 10 mM oxalate. 10 mM oxalate also blocked the formation of 32P-labeled sph-1-P indicating that it is an inhibitor of sph-1-P formation. The studies establish that the ER membrane contains the necessary kinase to convert sphingosine to sph-1-P which functions as a powerful mediator of

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Line; Cricetinae; Endoplasmic Reticulum; Heparin; Inositol 1,4,5-Trisphosphate; Kinetics; Lysophospholipids; Microsomes; Models, Biological; Muscle, Smooth; Oxalates; Oxalic Acid; Signal Transduction; Sphingosine